The agent used most widely to treat women with breast cancer is the triphenylethylene antiestrogen, trans-tamoxifen (TAM), which continues to undergo extended trials as an adjuvant therapy in estrogen receptor (ER) positive pre- and post-menopausal patients. Although it is known that TAM interferes with hormonal induction of specific genes, the exact biochemical mechanisms by which this occurs are not understood. Transcription is enhanced after estradiol (E2) and a dimeric ER protein form a complex at a specific site on DNA, called an estrogen responsive element (ERE), near the promoter of a responsive gene. 4- Hydroxytamoxifen (4-OHT), an active metabolite of TAM, binds to the ligand site of ER, but as results from our laboratory and others suggest, the conformation of the ER is altered compared to that of E2-ER. One of our observations is that at saturation, the apparent molar amount of 4- OHT-ER that can bind to either isolated cell nuclei, or plasmids bearing EREs, is only about half that of E2-ER. Among the explanations that we will evaluate is that a conformational change induced upon 4-OHT-ER-ERE complex formation, allows binding of only one, instead of the normal two, ligands per ER dimer. When several ERE sequences are in tandem, E2-ER binds cooperatively, but 4-OHT-ER does not, indicating that the conformational alteration in ER influences ER-ER interaction during DNA binding. Preliminary results show that antibodies to the DNA binding domain of ER can differentially inhibit E2-ER vs. 4-OHT-ER binding, revealing details of the conformational change. Additional results demonstrate that 4-OHT-ER dissociates more slowly from EREs than E2-ER. Further information will be obtained by DNA footprint analysis. The role of ERE sequence, spacing and stereoalignment on ER-DNA binding parameters will also be determined. The effect of these changes in EREs on promoter induction will be evaluated by transfection analyses. Overall, results will reveal structural and mechanistic consequences of 4-OHT binding to the ER.